Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific arginine vasopressin (AVP) binding sites were identified and characterized using Leydig cell membranes prepared from a clonal murine Leydig-derived cell line, TM3. 3H-AVP binding data analyses demonstrated that the radioligand binds to a high affinity, low capacity, homogeneous class of sites with a dissociation constant of 0.5 nM. Characterization of these AVP binding sites included competition studies. Displacement of 3H-AVP binding with high affinity by unlabelled AVP, LVP and the V1 antagonist, d(CH2)5Tyr(Me)AVP, indicated that the Leydig cell AVP receptor is of the V1 type. Furthermore, AVP did not increase adenylate cyclase activity in TM3 membranes, a finding consistent with the V1 type of AVP receptor. No competition with 3H-AVP was found with the V2 agonist, dVDAVP, or the selective oxytocin agonist, [Thr4,Gly7]oxytocin. No specific binding for oxytocin was found in Leydig cell membranes. No specific binding for either 3H-AVP or 3H-oxytocin was observed in membranes prepared from the Sertoli cell line or peritubular cell line. These findings indicate that murine Leydig cells have specific AVP binding sites of the V1 type. These AVP sites are not coupled to the adenylate cyclase system.
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PMID:Identification and characterization of arginine vasopressin receptors in the clonal murine Leydig-derived TM3 cell line. 271 74

The LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells in vitro has been investigated using sub-physiological to pharmacological concentrations of LH. Leydig cells pretreated with LH for 1 h in the presence of a phosphodiesterase inhibitor showed a dose-dependent decrease in the subsequent response to a high dose of LH; this was significant with concentrations of greater than 350 fmol/l. The maximum amount of desensitization was 60%. The time-course of LH-induced desensitization of the adenylate cyclase system was investigated; with 3 nmol LH/l cyclic AMP was increased in a linear manner up to 45 min, after which time there was either no further production or (with the higher concentrations) a decreased rate of production. In order to determine whether the 'non-desensitized' adenylate cyclase activity was dependent upon LH, the LH-treated cells were acid-washed to remove residual LH; cyclic AMP production still continued, albeit at a lower rate, thus indicating that this adenylate cyclase activity did not require the further presence of LH. The effect of various concentrations of LH on the level of surface-associated LH receptors was determined in the presence of monensin to prevent receptor recycling. A dose- and time-dependent decrease was found; this was significant after 2 h with 3.5 pmol LH/l and reached a maximum of 63% of the control with 3.5 nmol/l. A dose- and time-dependent reversal of desensitization occurred if the cells pretreated with LH were washed and reincubated; with 35 pmol LH/l and after 2 h the cells were fully responsive to a challenge with fresh LH. With higher concentrations of LH in the pretreatment, partial or no recovery was found. These studies demonstrate that physiological and sub-physiological concentrations of LH induce a rapid desensitization of Leydig cell adenylate cyclase. Internalization of occupied and unoccupied LH receptors also occurred. It is proposed that these two events are linked and may, paradoxically, and because of the low numbers of LH receptors, be necessary to maintain the normal response of Leydig cells to LH in vivo.
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PMID:The dynamics of LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells at physiological levels of LH. 282 28

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.
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PMID:Multiple regulation of testicular steroidogenesis. 282 90

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.
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PMID:Multistep regulation of Leydig cell function. 282 91

The mechanism by which luteinizing hormone (LH) promotes the production of testosterone in Leydig cells by binding to its high affinity sites was reinvestigated. Collagenase dispersed interstitial cells when purified by the application of a variety of techniques such as unit gravity sedimentation, gradient centrifugation, and a combination of the two procedures, were separated into two LH/hCG responsive cell fractions. The two types of interstitial cells displayed distinct biochemical and morphological characteristics. One cell type (the light cell) bound 125I-labeled human chorionic gonadotropin (125I-labeled hCG) with high affinity (Ka approximately equal to 3.33 x 10(9) M-1) but testosterone was not produced by this cell type as a result of hCG target cell receptor interaction. On the other hand, hCG stimulated the production of testosterone in another cell type (the dark/heavier cell). Steroidogenesis was maximally stimulated (700-800 percent over basal) by concentrations of hCG in the range of 3 x 10(-10) M, but high affinity binding sites for 125I-labeled hCG were not detectable. The residual binding that occurred did not obey saturation kinetics and was predominantly nonspecific. The stimulation of steroidogenesis by hCG in dark/heavier cells was dose and time dependent. Addition of dibutyryl or bromo cAMP (1 mM) to the cell suspension resulted in production of testosterone demonstrating the involvement of an hCG sensitive adenylate cyclase system in the transfer signaling process. These observations suggest the lack of a direct association between the occupancy of high affinity binding sites by hCG and testosterone production in rat Leydig cells. The stimulation of a biological response by a pathway independent of hCG occupancy of high affinity binding sites on Leydig cell is discussed and morphology of light and dark/heavier cells is presented. Autoradiographic evidence substantiates the conclusions.
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PMID:Demonstration of hCG binding sites and hCG stimulated steroidogenesis in different populations of interstitial cells. 282 79

Rat Leydig cells possess functional high-affinity receptors for angiotensin II (AII). AII inhibits adenylate cyclase activity in Leydig cell membranes and reduces basal and human chorionic gonadotropin (hCG)-stimulated cAMP pools and testosterone production in intact cells. Treatment of cells with an inhibitory dose of forskolin (10(-9) M) and a submaximal dose of AII caused additive inhibition of hCG-stimulated events. The inhibitory action of AII was largely prevented by pertussis toxin prior to the addition of AII alone or in the presence of hCG. This study and our recent report on inhibitory action of low doses of forskolin, 10(-12)-10(-9) M (Khanum, A., and Dufau, M.L. (1986) J. Biol. Chem. 261, 11456-11459) are indicative of a pertussis toxin-sensitive subunit of adenylate cyclase available for acute regulation of Leydig cell function. 8-bromo-cAMP bypasses the inhibitory effect of forskolin as well as AII. We have, therefore, demonstrated functional AII high-affinity receptor and an acute inhibitory effect of AII on hCG action in Leydig cells. Our results have provided evidence for a pertussis toxin-sensitive guanine nucleotide inhibitory protein as mediator of the effect of AII. These findings further emphasized the importance of the cAMP pathway in the Leydig cells, and studies also suggest that tubular and locally produced AII could negatively modulate luteinizing hormone stimulation of Leydig cells.
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PMID:Angiotensin II receptors and inhibitory actions in Leydig cells. 283 94

Modifications of carbohydrate structures of hCG, such as deglycosylation or desialylation, have been shown to reduce the biological activity of the hormone derivatives in vivo. We posed the question of whether deglycosylated hCG (dg-hCG) and desialylated hCG (ds-hCG) would behave as agonists at the LH/CG receptor in the primate in vivo, as this would bear on their potential clinical utility as LH/CG agonists or antagonists. Thus, we administered large doses (approximately 3 nmol) of highly purified dg-hCG, ds-hCG, hCG, or normal saline as a rapid iv injection to adult male cynomolgus monkeys (n = 3/group). Mean areas under the curves of plasma T over the first 6 h achieved with dg-hCG and ds-hCG were about 5-fold, significantly (P less than 0.05) greater than that in the saline controls and not significantly (P greater than 0.05) different from that in hCG-injected animals. Despite comparable plasma T responses in the first 6 h, mean plasma concentrations of ds-hCG, dg-hCG, and hCG differed dramatically among the groups. Plasma ds-hCG and dg-hCG levels were undetectable by 15 and 180 min, respectively, while the mean plasma hCG level was more than 2.10 nmol/L at 360 min. These data indicate that 1) dg-hCG is a full agonist at the LH/CG receptor in the primate in vivo, despite having minimal intrinsic activity in the rat Leydig cell adenyl cyclase assay and being able to near-completely antagonize hCG action therein; and 2) ds-hCG is a full agonist in the monkey in vivo, capable of stimulating a full testicular response over 6 h, despite being cleared from the circulation in 15 min. We conclude that the signal transduction system at the monkey LH/CG receptor is capable of achieving full steroidogenesis despite dramatically shortened exposure to stimulus or exposure to a stimulus with markedly reduced adenyl cyclase-stimulating activity in vitro.
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PMID:Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin. 290 63

Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were evaluated by using purified adult Leydig cells from Sprague-Dawley rats. Purified Leydig cells were found to contain both high and low affinity binding sites for insulin, with Ka values of 1.08 X 10(9) and 1.1 X 10(7) M-1, respectively. Using affinity cross-linking of [125I]iodoinsulin to plasma membrane insulin receptor, several bands were identified by autoradiography under nonreduced conditions with mol wt of 230,000, 280,000, and 300,000. After reduction with 50 mM dithiothreitol, only one band was identified with a mol wt of 130,000, consistent with the alpha-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 X 10(9) M-1. Multiple high mol wt bands (greater than 250,000) were identified under nonreduced conditions by affinity cross-linking. Under reduced conditions, one band with an approximate mol wt of 135,000 was identified. Purified Leydig cells (10(5) cells/ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 Nutrient Mixture (1:1) containing 0.1% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2-95% air. Insulin and IGF-I stimulated testosterone formation as early as 3 h after administration, and their effects were completely blocked by the addition of a protein synthesis inhibitor, cycloheximide (1 microgram/ml). Insulin and IGF-I also significantly potentiated hCG-and 8-bromo-cAMP-induced testosterone formation. Furthermore, insulin and IGF-I potentiated hCG-stimulated cAMP formation. This suggests that insulin and IGF-I have effects at both the LH receptor sites and the steps beyond adenylate cyclase. The ED50 values of insulin and IGF-I-stimulated testosterone formation were comparable (25 ng/ml). In conclusion, we found that Leydig cells contain specific insulin and type I IGF receptors, and both insulin and IGF-I are capable of modulating Leydig cell steroidogenesis.
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PMID:Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture: a comparative study. 294 38

When the phorbol ester, 4 beta-phorbol-12-myristate-13-acetate (PMA) or bacterial phospholipase C (PL-C) is added to a preparation of purified adult rat Leydig cells, containing 2 mM CaCl2, a time- and dose-dependent decreases of LH-stimulated testosterone production is observed. After a 3 h stimulation with oLH (100 ng/ml), PMA (100 ng/ml) and PL-C (1.6 U/ml) do not affect the cell viability or the hCG specific binding, while cAMP accumulation is significantly reduced; cAMP-stimulated steroidogenesis is diminished only in the presence of PL-C. These observations suggest that in vitro: (i) activated Ca2+- and phospholipid-dependent protein kinase is implicated in the regulation of rat Leydig cell steroidogenesis by LH at a step before the adenylate cyclase; (ii) phospholipids play an important role in cAMP-stimulated testosterone synthesis.
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PMID:Effect of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells. 299 25

PTH receptor-stimulating proteins may be a common mediator of humoral hypercalcemia of malignancy (HHM). Such proteins exhibit adenylate cyclase-stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification. Acid/urea tumor extracts from a murine squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34) amide. ACSA from murine tumor extracts has now been further purified using solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating protein extracted from rat Leydig cell and human HHM tumors. The ACSA in these three peaks of murine tumor extract elutes in the same region as human tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat osteosarcoma (ROS) adenylate cyclase assays. The remaining half of the ACSA from murine tumor extracts elutes as a single peak (peak II) at a higher acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine tumor acid/urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct protein, not previously reported in tumor extracts, that may act as a postreceptor step in the adenylate cyclase system.
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PMID:Two species of adenylate cyclase-stimulating activity in a murine squamous carcinoma model of humoral hypercalcemia of malignancy. 300 44


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